Melanoma antigen gene protein-A11 (MAGE-11) is a novel androgen receptor (AR) coregulator that interacts with the AR NH2-terminal FXXLF motif and increases AR transcriptional activity through mechanisms that require phosphorylation and monoubiquitinylation. The post-translational modification of MAGE-11 occurs in response to epidermal growth factor signaling and appears to involve cell cycle regulatory proteins. Our studies will determine the mechanisms that underlie the MAGE-11 dependent increase in AR transcriptional activity. Aim 1 will pursue preliminary evidence that MAGE-11 interacts directly with SRC/p160 and p300 transcriptional coregulators. MAGE-11 shares sequence similarity with the early adenoviral protein E1A, and like E1A, appears to interact directly with p300. We will identify the interaction sites between MAGE-11, p300 and SRC/p160 as a step toward understanding cell signaling targets that modulate AR activation function 1 in the NH2-terminal domain. Aim 2 pursues evidence for a phosphorylation-dependent interaction between MAGE-11 and the F-box protein Skp2 (S phase kinase-associated protein 2) to test our hypothesis that MAGE-11 functions as a subunit of the SCF (Skp1-cullin-Skp2 F-box) E3 ubiquitin ligase complex. We will determine whether MAGE-11 is a component of a cullin-based E3 ligase complex that provides a link between AR and its transcriptional coactivators. We will determine the function of CHIP (carboxyl-terminus of Hsp70-interacting protein) in the SCF ligase complex and identify and characterize the E3 ligase that ubiquitinates MAGE-11, a modification required for AR coregulator function. Our studies will provide insight into mechanisms whereby AR regulates gene transcription in human cells.